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Single Image-Based Vignetting Correction for Improving the Consistency of Neural Activity Analysis in 2-Photon Functional Microscopy.

ABSTRACT: High-resolution functional 2-photon microscopy of neural activity is a cornerstone technique in current neuroscience, enabling, for instance, the image-based analysis of relations of the organization of local neuron populations and their temporal neural activity patterns. Interpreting local image intensity as a direct quantitative measure of neural activity presumes, however, a consistent within- and across-image relationship between the image intensity and neural activity, which may be subject to interference by illumination artifacts. In particular, the so-called vignetting artifact-the decrease of image intensity toward the edges of an image-is, at the moment, widely neglected in the context of functional microscopy analyses of neural activity, but potentially introduces a substantial center-periphery bias of derived functional measures. In the present report, we propose a straightforward protocol for single image-based vignetting correction. Using immediate-early gene-based 2-photon microscopic neural image data of the mouse brain, we show the necessity of correcting both image brightness and contrast to improve within- and across-image intensity consistency and demonstrate the plausibility of the resulting functional data.

SUBMITTER: Li D

PROVIDER: S-EPMC8766855 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Significance: Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. Aim: We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging. Approach: We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity. Results: First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics. Conclusions: The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.

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Single Image-Based Vignetting Correction for Improving the Consistency of Neural Activity Analysis in 2-Photon Functional Microscopy.

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